Host–bacteria interactions: ecological and evolutionary insights from ancient, professional endosymbionts

Abstract Interactions between eukaryotic hosts and their bacterial symbionts drive key ecological and evolutionary processes, from regulating ecosystems to the evolution of complex molecular machines and processes. Over time, endosymbionts generally evolve reduced genomes, and their relationship with their host tends to stabilize. However, host–bacteria relationships may be heavily influenced by environmental changes. Here, we review these effects on one of the most ancient and diverse endosymbiotic groups, formed by—among others—Legionellales, Francisellaceae, and Piscirickettsiaceae. This group is referred to as Deep-branching Intracellular Gammaproteobacteria (DIG), whose last common ancestor presumably emerged about 2 Ga ago. We show that DIGs are globally distributed, but generally at very low abundance, and are mainly identified in aquatic biomes. Most DIGs harbour a type IVB secretion system, critical for host-adaptation, but its structure and composition vary. Finally, we review the different types of microbial interactions that can occur in diverse environments, with direct or indirect effects on DIG populations. The increased use of omics technologies on environmental samples will allow a better understanding of host–bacterial interactions and help unravel the definition of DIGs as a group from an ecological, molecular, and evolutionary perspective.


Introduction
Div erse or ganisms liv e together, and by doing so become mor e than they are as indi viduals: the y form symbioses (Bary 1879 ).
Symbioses ar e div erse, ubiquitous in natur e, and r epr esent major driving forces of evolutionary change (Bennett andMoran 2015 , Oliver andRussell 2016 ).They create conditions favourable to the emergence of complex life forms along the mutualism-parasitism continuum, allowing for expansion into pr e viousl y inaccessible ecological niches (Toft and Andersson 2010, Skelton et al. 2016, Sudakaran et al. 2017 ).Interactions along the mutualism-parasitism continuum include (i) m utualism, wher e or ganisms jointl y cooperate, (ii) commensalism, where one partner benefits with no effect to the other, and (iii) par asitism, wher e onl y one of the partners benefits at the expense of the other (Parmentier and Michel 2013 ).Symbionts can be either intracellular (endosymbionts), extracellular (ectosymbionts), or can be tightly associated with the host (such as episymbionts).Endosymbionts, which are the focus of this r e vie w, can be facultativ e or obligate, depending on whether the symbionts can survive extended periods of time outside of their host (Toft and Andersson 2010 ).By definition, endosymbionts live intracellularly within a host and may reside in the cytoplasm, inside vacuoles, or e v en inside the host nucleus or mitochondria.
Endosymbioses are at the origin of some of the most important eukaryotic inno vations .It is widel y accepted that mitoc hondria and plastids, in early eukary otes, w er e deriv ed fr om an alpha pr oteobacterial endosymbiont and a c y anobacterial endosymbiont, r espectiv el y (Sa gan 1967 , Huang and Gogarten 2007, Gray 2012, Martijn et al. 2018, Sibbald and Archibald 2020 ).Besides these, many other bacterial groups have been associated with eukaryotes, some of them for over a billion years (Toft and Andersson 2010 ).The most diverse, successful, and ancient endosymbiont linea ges closel y associated with eukaryotes ar e found within Alphaproteobacteria , Gammaproteobacteria , and Chlamydiae (Köstlbacher et al. 2021, Wang and Luo 2021, Hugoson et al. 2022 ).Members of these lineages can be described as ancient professional endosymbionts (Husnik et al. 2021 ) because they have established long-term symbiotic relationships, allowing them to accum ulate intricate ada ptations with their hosts thr ough coe volution over hundreds of millions of years.Endosymbionts interact with their hosts in mutualistic, commensal, or pathogenic manners, and include pathogens that are significant causes of disease in human, liv estoc k and crop (Duron et al. 2018a, Dharamshi 2021, Schön et al. 2022 ).Specific molecular mechanisms are welldescribed for certain ancient professional endosymbionts, mostly within Alphaproteobacteria and Chlam ydiae .Indeed, Ric kettsiales use se v er al known factors to interact with their hosts, including a type IV secretion system, effector proteins that manipulate the host cell, and an Adenosine triphosphate (ATP) / Adenosine diphosphate (ADP) tr anslocase, whic h facilitates ener gy par asitism by exchanging ATP for endogenous ADP from the host cell (Schön et al. 2022 ).In gener al, secr etion systems ar e crucial to hostsymbiont interactions across the mutualism-parasitism continuum, spread by horizontal transfer and evolve through co-option, neofunctionalization, speciation, and de novo acquisition of single or multiple proteins at a time (Denise et al. 2020 ).Secretion systems , thus , pro vide endosymbionts with various specific functions , e .g. facilitating cell adhesion, injecting effectors and virulence factors during host infection, or excreting toxic compounds (Segal et al. 2005, Costa et al. 2015 ).The study of ancient endosymbionts and their host-interaction systems is invaluable for elucidating the evolutionary processes and the specific molecular and ecological mechanisms underlying host-endosymbiont interactions, along the m utualism-par asitism continuum.Some endosymbiont gr oups, suc h as the ones mentioned abov e, ar e wellr e vie wed (Köstlbac her et al. 2021, Wang and Luo 2021, Castelli et al. 2022, Dharamshi et al. 2023 ) but m uc h less is known about the-potentiall y-oldest one, whic h we r efer to as the Deepbr anc hing Intr acellular Gammaproteobacteria (DIG).
DIGs are defined as the Legionellales , tr aditionall y divided in two families, Legionellaceae and Coxiellaceae , and se v er al r elated intr acellular groups .T he Legionellales order harbours a wide ecological div ersity of gener a with: (i) facultativ e intr acellular pathogens of amoebae such as Aquicella and Legionella ( Legionella are also accidental human pathogens, causing Legionnaires' disease); (ii) obligate intr acellular pathogens, suc h as Coxiella burnetii , the a gent of Q-fe v er; (iii) facultativ e intr acellular symbionts of arthr opods such as ' Ca.Rickettsiella viridis (Tsuchida et al. 2010 ), and (iv) e v en an obligate mutualistic endosymbiont of a ciliate, in which the endosymbiont has replaced the mitochondrion (Graf et al. 2021 ).Beyond Legionellales , DIGs also include Francisellaceae (including mammalian pathogens like F. tularensis ), Fastidiosibacteraceae (such as Paramecium endosymbionts from marine seawater envir onment), and Pisciric kettsiaceae (containing so far onl y one intr acellular genus, Pisciric kettsia , a fish pathogen) (Duron et al. 2018b, Barril 2022, Xiao et al. 2018, F ry er and Hedrick 2003 ).These groups , named abo ve as in the traditional taxonomies (e.g. at NCBI or through LPSN) (Parte et al. 2020 ), have been reclassified as orders in the genome-based taxonomy de v eloped by Genome Taxonomy Database (GTDB) (Parks et al. 2022 ).Hereafter and where v er possible, we will adopt the latter classification (Table 1 ), among others because the GTDB tree shows the main DIG classes as monophyletic (Fig. 1 ).Furthermore, one of the landmarks of DIGs (although there are notable exce ptions, lik e the Francisella genus) is the possession of a type IVB secretion system (T4BSS).This secretion system facilitates symbiotic interactions all across the par asite-m utualism continuum, by injecting effector pr oteins into the host cytoplasm (Costa et al. 2015, Hugoson et al. 2022 ).
This article r e vie ws host-bacteria inter actions, with a focus on ecological and evolutionary insights from DIG bacteria.The environmental distribution of these ancient professional bacterial endosymbionts is r e vie wed in section -Environmental distribution of DIGs, along with their diversity in different biomes.Their molecular features are then discussed in section -Impact of molecular mechanisms on the mutualism-parasitism continuum of DIGs, including their impact on the placement along the m utualistic-pathogenic continuum.Finall y, the inter actions between endosymbionts and other micr oor ganisms pr esent in their host ar e r eported in section -Influence of the microbiotainteraction with other bacteria and endosymbionts.

Envir onmental distrib ution of DIGs
Growing DIGs in laboratory settings is difficult, due to their intracellular lifestyle and dependence on their host (Table 1 ).Curr entl y, out of the potential hundreds of genera of DIGs (Graells et al. 2018 ), only species of the genera Legionella , Aquicella , Coxiella , Piscirickettsia , Fangia , and Francisella can be grown axenically (Santos et al. 2003, Omsland 2012, Martínez et al. 2018, Xiao et al. 2018, Vallesi et al. 2019 ).Fastidious organisms often cannot be sequenced with traditional genomic techniques, making metagenomics the method of choice for exploring their div ersity.A pr evious study, which examined large amounts of metabarcoding data, found that Legionellales are diverse and ubiquitous (both geogr a phicall y and envir onmentall y), but at v ery low abundance (about 0.1%) (Graells et al. 2018 ).Shotgun metagenomics is providing a better view of the distribution, diversity, and functional potential of DIGs.Analysis of DIGs through metagenomic datasets are crucial to better understanding potential vectors for emerging pathogens in relation to global environmental changes (Ranjan et al. 2016 ).
In this section, we surveyed the geogr a phic and environmental distribution of DIGs, r e vie wing publicl y av ailable meta genomic data.Briefly, data and metadata were collected for all samples present on MGnify using the available Application Programming Interface (API) and samples with at least one 16S rRNA read attributed to DIGs were retained for analysis ( n = 5605) (Gurbich et al. 2023 ).To test a more conservative approach, cut-offs of 3 reads ( Supplementary Figs 1 -4 ) and 10 reads were applied and the number of positive samples decreased ( −27% and −53%, respectiv el y).As most DIGs ar e r ar e (Gr aells et al. 2018 ), suc h a decrease is expected with a 3-, r espectiv el y 10-fold increase in the cut-off.Div ersity, geogr a phic distribution, and the effect of envir onmental factors (temper atur e, depth, and pH) on DIG abundance wer e gather ed and visualized using Python, including the pac ka ge mgtoolkit, and R, including the pac ka ges ggplot2, tidyv erse, ggpubr, ma ptools, ggsn, scales, and phyloseq (Van Rossum and Dr ake 2009 , Baquer o 2019 , R Cor e Team 2022 , Wic kham et al. 2022, Bivand et al. 2023, Kassambara 2023, McMurdie and Holmes 2023, Ranathunga 2023, Wickham and RStudio 2023, Wickham et al. 2023 ).

Geographical distribution
DIGs ar e globall y distributed acr oss the world, with a few exceptions (Fig. 3 ).This is particularly true for the Coxiellaceae , Legionellaceae , and Piscirickettsiaceae : all continents harbour at least one sequence affiliated to them.This distribution suggests that these families are generalist taxa, i.e. taxa with a cosmopolitan distribution and metabolically active according to their level of tolerance to environmental conditions (Kilroy et al. 2007, Cox et al. 2019 ).The Legionellaceae and Coxiellaceae datasets are dominated b y tw o types of genera, Legionella and Coxiella ( > 65%).These tw o gener a ar e well-r epr esented in the databases, and ar e r esponsible for major public health problems on five continents (Celina andCerný 2022 , Lockwood et al. 2022 ).Cases of Legionnaires' disease and Q fe v er hav e shar pl y incr eased ov er the last two decades, with an 8-10-fold increase of Legionnaires' disease in Europe and a 2-3-fold increase of Q fever in the United States (Committee on Management of Legionella in Water System 2019 , CDC 2021 ).In gener al, amoebae and tic ks-both globall y distributed-ar e the most w ell kno wn hosts for the Legionellaceae and Coxiellaceae , respectiv el y (Dur on et al. 2017 , Celina andCerný 2022 ).Unsurprisingly, Legionellaceae and Coxiellaceae are the most geographically widespr ead families, pr esumabl y due to the global distribution of  their hosts and the increased outbreak monitoring and tracking (Felice et al. 2019 ).
Piscirickettsiaceae is also widely distributed (Fig. 3 D), with a high concentration of samples that were found in the Atlantic and P acific Oceans.Man y salmon farms ar e located in these oceans, which is a likely explanation for the presence of reads attributed to Piscirickettsia salmonis , the agent of piscirickettsiosis (an infectious salmon disease) (Rozas-Serri 2022 ).
Fastidiosibacteraceae is globally distributed, although to a lesser degree than the former families (Fig. 3 F).They are not found close to the poles (Arctic and Antarctic).Mor eov er, compar ed to the other DIG families, ther e ar e fe wer positiv e samples in the Southern Hemisphere ( < 35%) (Fig. 3 F).Only five described gener a ar e part of this famil y, and all originate fr om aquatic envir onments fr om fiv e continents (e.g.Fangia hongkongensis isolated from Asia) (Xiao et al. 2018 ).In addition, Caedibacter taeniospiralis r epr oduces in the globally distributed protist Paramecium caudatum (Beier et al. 2002, Krenek et al. 2012 ).Ho w e v er, the lo w er abundance of this family in our dataset is likely due to the lack of documentation and the small number of species it contains, and is unlikely to represent the reality of its geographical distribution.
Francisellaceae is also distributed worldwide, with an especially important presence in salt water en vironments , particularly in the Atlantic and Pacific Oceans (Fig. 3 E).Several Francisellaceae species , e .g. F rancisella noatunensis (Colquhoun and Duodu 2011 ), F. piscicida (Ottem et al. 2008 ) are fish pathogens, while others, e.g.F. salimarina (Li et al. 2020 ) and Pseudofrancisella aestuarii (Zheng et al. 2019 ) were isolated from estuaries.Among those found on land, the species F. tularensis , responsible for the zoonosis Figure 2. Global distribution of metagenomic samples from public databases and sample distribution across DIG families.MGnify has used different taxonomies in the different versions of its pipeline, but most analyses were performed with the SILVA database (prior version 138), which mostly reflects the traditional taxonomy.At family level, the discrepancies are minor, but the genera from Fastidiosibacteraceae are included in Francisellaceae in the GTDB taxonomy.tular emia, is spr ead ov er long distances by its hosts, suc h as tic ks and mosquitoes (Lundström et al. 2011, Hennebique et al. 2019 ).

Biomes
DIGs ar e pr esent in all major biome categories, as defined by the ontology from the Earth Microbiome project (EMP ontology) (Thompson et al. 2017 ), in v arying pr oportions.As expected, water samples displayed the highest share of positive samples (43%), but other biomes also had a high proportion of samples positive for DIGs: animal corpus (mostly gut samples), 10%; soil, 4.9%; and sediments, 4.6%.A total of 10% of the biomes that could not be classified as above were also positive for DIGs.(Fig. 4 A).
Among all samples where DIG families are present, about half (49.5% on av er a ge ov er the fiv e families) ar e , on a v er a ge, fr om the aquatic biome (Fig. 4 ).Despite being present in many aquatic samples, DIGs are mostly there in low abundance, less than 0.01%.Of the more abundant DIG famil y taxa, Pisciric kettsiaceae and Legionnnellaceae are found in higher abundance in the latter ecosystems.While fr equentl y identified in freshwater or soil samples, or within their hosts, Coxiellaceae , Legionellaceae , Piscirickettsiaceae , and F rancisellaceae ha v e been, mor e r ecentl y, also found in salt waters (Tsao et al. 2017, Hastuti et al. 2022, Bergman et al. 2023, Eriksson et al. 2023 ).T he genus F rancisella ( F rancisellaceae ) is found in extr eme aquatic ecosystems, suc h as deep-sea hydr othermal v ents in the Pacific and Atlantic oceans, where it can play a role in sulphur oxidation (Sylvan et al. 2012, Ding et al. 2017 ).Certain micr oor ganisms of the famil y Pisciric kettsiaceae ar e commonl y found in hypersaline, sulphur-rich, or soda lakes (gener all y at low abundances < 0.5%), supporting the hypothesis of an ecological role for these organisms in sulphur and methanogenesis cycles (Borin et al. 2009, Paul et al. 2016 ).The Fastidiosibacteraceae famil y is poorl y studied, and all isolates curr entl y av ailable in databases ar e exclusiv el y fr om lake ecosystems (Xiao et al. 2018, Salam et al. 2019 ); for example the species Fastidiosibacter lacustris was isolated from a water sample from Lake Haizhu in China (Xiao et al. 2018, Brunet et al. 2021, Da vido vich et al. 2022 ).Se wa ge tr eatment plants ar e incr easingl y consider ed as sources of pathogens and species of Legionellaceae and Coxiellaceae have been identified in these envir onments (Sc hets et al. 2013, v an den Ber g et al. 2023 ).In gener al, aer obic biological water treatment systems provide an optimal environment for the growth of these micr oor ganisms due to high concentrations of organic nitrogen and oxygen, ideal temper atur es, and the pr esence of pr otozoa (Caicedo et al. 2019 ).The survival of some of these endosymbionts in aquatic ecosystems may be favoured by their hosts; indeed, amoebae are common hosts for Legionellaceae , Coxiellaceae , and Francisellaceae (Duron et al. 2018a, Hennebique et al. 2019, Price and Abu Kwaik 2021, Solbach et al. 2021 ).In 2021, a stud y look ed in detail at the longterm interactions of the tularemia pathogens F. tularensis subsp .holarctica , F. novicida , or F. philomiragi a with amoebae of the Acanthamoeba species and sho w ed that amoebae are likely to promote the survival of Francisella in aquatic environments, including the tularemia pathogen F. tularensis (Hennebique et al. 2021 ).
Se v er al DIGs hav e aquatic metazoans as hosts, whic h may explain their high pr e v alence both in aquatic and animal gut biome categories (on av er a ge 8.8%, Fig. 4 ).It is particularl y a ppar ent for Francisellaceae , present in 26% of the animal gut samples (Fig. 4 E).This might be explained by the r ecurr ent isolation of F. tularensis from the digestive systems of mosquitoes , bedbugs , lice , flies , and ticks (Telford and Goethert 2020 ).Francisella tularensis survives the harsh, acidic condition found in the insect gut tr acts, mostl y by inv ading erythr ocytes that tic ks ingest during a blood meal (Sc hmitt et al. 2017 ).In addition to protection from low pH conditions, erythr ocytes pr ovide haemoglobin, whic h can help counter act oxidativ e str ess , and heme , whic h serv es as an ir on source necessary for F. tularensis survival.The high prevalence of Piscirickettsiaceae and Coxiellaceae both in aquatic and animal gut biome categories might also be caused by their metazoan hosts.Piscirickettsia salmonis causes piscirickettsiosis, a disease affecting farmed salmon, trout and sea bass.Piscirickettsiosis is a systemic infection characterized by colonization of multiple organs including the kidney, liver, spleen, intestine, brain, ovary, and gills of salmon (Aravena et al. 2020 ), typically resulting in mortality rates between 10% and 30%.In addition to ticks, some Coxiellaceae species also colonize the gut of other metazoans (3.8% of samples, Fig. 4 B).For example, Coxiella cheraxi , causes rickettsiosis in the red-legged crayfish ( Cherax quadricarinatus ).Coxiella cheraxi can colonize the midgut of its host and cause 22%-80% mortality in natur all y infected crayfish (Da vido vich et al. 2022 ).
Sediment samples accounted for an av er a ge of 6% of the samples positive for DIGs (Fig. 4 ).For Piscirickettsiaceae and Fastidiosibacteraceae , it amounted to 10.6% and 8.0% of samples, r espectiv el y (Fig. 4 D and F).Se v er al studies hav e identified Pisciric kettsiaceae in a variety of sediments, including coastal, ri verine, dee p, and estuarine sediments (Cleary et al. 2017, Xie et al. 2018, Sonthiphand et al. 2023 ).Fastidiosibacteraceae ar e gener all y isolated fr om lake biomes, where sediment samples are collected, which partly explains their high pr e v alence in this biome type.During sedimentation and the passage of o verla ying water at the sediment-water interface, the number of bacteria increased by 3-5 orders of mag-nitude, perhaps making them easier to detect in this biome (Wetzel 2001 ).Amoebae, a common host of Coxiellaceae and Legionellaceae , are identified in most biomes, including sediments.It may explain why 4.9% and 7.1% of samples from this biome category ar e positiv e for theses two families, r espectiv el y: e.g.Legionella ar e found in tank sediments, (Lu et al. 2015 ) and Coxiella in water plant system sediments, (Corsaro et al. 2010 ).The DIGs are also found in sediments from mines and anthropized ca ves .Indeed, in subsurface environments such as caves and mines, the Proteobacteria phylum is the second-most abundant, with e.g.Coxiellaceae identified in sediments from a cave in Thailand (1.5% of the microbial community) (Leon et al. 2018, Wiseschart et al. 2019, Bontemps et al. 2022 ).Coxiella burnetii was identified in a cave in Spain, where it caused a Q fe v er outbr eak in 2021 (five cases) (Hurtado et al. 2023 ).Caves can be considered as a reservoir for pathogens, where Legionella ar e r egularl y identified (e .g. 3.3% in Bossea Ca ve , Italy) (Biagioli et al. 2023 ).The presence of Legionella is often associated with amoebae, whic h ar e gener all y pr esent in cav es.Indeed, the phylum Amoebozoa is often found in soil and water, a c har acteristic shared by karst environments (Alonso et al. 2019, Bontemps et al. 2022 ).Over nine species of Legionella associated with amoebae have been identified in Lascaux cave (among others L. pneumophila , L. jordanis , and L. oakridgensis ) (Bastian et al. 2009 ).Like amoebae, host ticks of certain Francisellaceae and Coxiellaceae are found in subterr anean envir onments with high humidity; this is the case for Agardsides and Carios ticks (Kazim et al. 2021 ).
The aerosols category represents only 2.1% of the samples positive for Legionellaceae , 0.3% and 0.1% for Coxiellaceae and Pisciric kettsiaceae , r espectiv el y (Fig. 4 B-D).The most common route of transmission of Legionella , in cases of legionellosis, is the As expected, DIGs are most often found in aquatic environments, follo w ed b y animal guts and sediments, but also, increasingly, in saline environments (Fig. 4 ).This ecological description remains incomplete, with 25% of DIG-positive samples (up to 42% for the Fastidiosibacteraceae ) having no associated biomes (Fig. 4 ).

Environmental factors
It is incr easingl y r ecognized that the emer gence, pr e v alence, and se v erity of diseases depend on complex inter actions between pathogens , hosts , and the en vironment (J ones et al. 2008(J ones et al. , Plowright et al. 2008 ) ). Se v er al risk factors, both biotic (age and species of the host) and abiotic (temper atur e, salinity, sunlight, and hydrodynamic connectivity), are known to contribute to the occurrence of epidemics and the alteration of microbial diversity in natur al envir onments.In this survey of the published metagenomic data, we recovered metadata for each metagenome available on MGnify (Gurbich et al. 2023 ), including depth, loca-tion, ele v ation, temper atur e , salinity, altitude , pH, latitude , longitude , nitrate , and oxygen concentration.Ho w ever, metadata is often patchy, and no single metagenome has all this information.Her e, we r e vie wed the effects of four environmental factors (temper atur e, depth, salinity, and pH) on the DIG community (Fig. 5 ).
Temper atur e is an important factor to consider, as rising global temper atur es can lead to changes in the abundance of certain micr oor ganisms and protists (Hosen et al. 2017 ).Despite the ease of measuring temper atur e, it is available only for a fraction of the samples, 28.7% of the data for Francisellaceae , 9.5% for Coxiellaceae and Legionellaceae , and 7.8% for Fastidiosibacteraceae (Fig. 5 A).Within these, ∼10% of the values are obviously erroneous (e.g.−9999.0• C or + 9999.9 • C), complicating downstr eam anal ysis, and highlighting the fact that too few users pr ovide corr ect metadata information when submitting sequences.For DIGs in general, the av er a ge temper atur e at whic h they ar e identified is 16.9 • C, with a range of −8 • C to 37 • C (Fig. 5 A).It is known that e.g.Legionella can m ultipl y at higher temper atur es (e.g.Tison et al. 1980, Kusnetsov et al. 1996 , Committee on Management of Legionella in Water Systems 2019 ).In Hot Springs National Park, Arkansas (USA), where hot spring water is piped at natur all y high temper atur es ( > 57 • C), five cases of legionellosis occurred in 2018-2019, indicating the presence of L. pneumophila bacteria in piped spring water (James et al. 2022 ).Conv ersel y, the pr esence of L. pneumophila has been reported in cold en vironments , such as in the freshwater lake of King George Island in the Antarctic Peninsula.Although the proportion of Legionella species is consistently low, while they have been fr equentl y detected e v en in Antar ctic lakes, sho wing their wide distribution in cold climates (Shimada et al. 2021 ).These results suggest that Legionella spp.adapted to extreme temperatures may be widely distributed in both low and high temperatur e envir onments.Temper atur e also affects the ability of DIGs to infect their hosts: for example, while at higher temper atur es L. pneumophila is able to parasitize A. castellanii cells, below 25 • C, A. castellanii has been observed to digest the L. pneumophila population infecting it (Moffat and Tompkins 1992 ).
In other DIG gr oups, temper atur e is a critical environmental factor in the de v elopment of outbr eaks.A study in salmon farms in Chile, suggests that temper atur e is positiv el y corr elated with fish mortality due to piscirickettsiosis, caused by Piscirickettsia spp.The observ ed pr e v alence of pisciric kettsiosis was ar ound 20% when the water temper atur e was below 9 • C, while it increased to 70% at temper atur es abov e 9 • C (Martínez et al. 2018, Br avo et al. 2020 ).Temper atur e is also a critical factor for francisellosis outbr eaks, wher e water temper atur e below 26 • C pr omotes the disease.A study conducted on Francisella shows that changes in temper atur e modulate the expression of o xidati v e str ess and heat shock genes, as well as metabolism-related genes .T he presence of DIG families over a wide range of temperatures ( −8 • C to 37 • C) is also associated with the host being able to ada pt mor e quic kl y to certain environmental conditions; ho w ever, temperature can affect both the host and its microbial partners (Lemoine et al. 2020 ).Indeed, thermal variations have a major impact on host metabolism, with extr eme temper atur es thr eatening surviv al and fertility.In addition, thermal stress can deplete obligate endosymbionts, destabilizing the association and reducing the fitness of both partners, thus limiting the host's viable temper atur e r ange (Zhang et al. 2019 ).A rise in temper atur e has been shown to cause a modulation in the expression of genes involved in the innate and ada ptiv e imm une r esponse in salmon and facilitate the de v elopment of P. salmonis (Martínez et al. 2018 ).
The most commonl y r eported metadata is the depth at which the samples were collected, with an average of 30.4% of the samples in our dataset containing this information, rising to 51.9% for Francisellaceae (Fig. 5 B).No study has dir ectl y compar ed the effects of depth (i.e. the effects of different depths in the same ecosystems) on the abundance and diversity of DIGs.Ho w e v er, it can be assumed that depth is a factor that affects the structure of micr obial comm unities, as it is associated with significant biotic and abiotic c hanges, suc h as the absence of light, CO 2 , oxygen, moisture, and nutrient availability (e.g.oligotrophy).For all the DIG families studied here, the depth data are similar and the av er a ge is around 0 m, meaning that most of the time they are identified when sampling at the surface, between the litter and mineral layer (Fig. 5 B).Certain DIG families are found on the seafloor (e.g.deep-sea hydrothermal vents, see section above).Although most DIG families are found in aquatic en vironments , very little information is curr entl y av ailable on their pr esence at differ ent le v els of the water column.
Man y studies hav e shown that salinity also alters the structure of the microbial community and reduces microbial activity.Salinity information is available for 11.6% of the DIG metagenomes, r anging fr om 0 to 61.3 pr actical salinity unit (PSU, gr am of salt per kilogram of water) for Fransicellaceae (Fig. 5 C).Indeed, a single Francisella species was detected by qPCR in waters ranging from 1 to 38 PSU (Brunet et al. 2021 ).The effect of salinity on the detection of Francisella sp. could not be e v aluated in that study, due to the insufficient number of saline and br ac kish water samples.Ho w e v er, Francisellaceae ar e found in fr esh, br ac kish, and saltwater, indicating that these bacteria can inhabit a wide range of water types (Fig 5 C).Similarly, Legionellaceae have been identified in natur al seawater fr om the Baltic Sea and the North Sea with salinities of 15-30 PSU (Linsak et al. 2021 ).Aquatic environments with low or moderate osmotic pressure are the main natural aquatic habitats for Legionella (Schwake et al. 2021 ).Although saltwater can cause envir onmental str ess to bacterial cells, the natur al pr esence of Legionella in this environment and their tolerance to it has long been r eported.Researc h on Legionella ecology in saline springs is limited and often assumes freshwater contamination.Although this is often the case, reports from high osmolality, isolated, and oceanic sites suggest that saline en vironments ma y also provide a natural habitat for Legionella (Schwake et al. 2021 ).Exposure of Legionella to high concentrations of sodium has previously been shown to inhibit the growth and virulence of laboratory strains and promote the growth of avirulent forms (Bergman et al. 2023 ).The ability of Legionella to survive and multiply in their protozoan hosts is mediated by the T4BSS, which is also involved in the sodium sensitivity phenotype observed in L. pneumophila .It has been postulated that sodium sensitivity is a result of leakage of sodium ions through the T4BSS (Bergman et al. 2023 ).It is ther efor e likel y that Legionella species hav e ada pted to surviv e in sodium-ric h envir onments, in the presence of sodium-resistant pr otozoa.Indeed, a pr e viousl y unidentified species, L. tunisiensis , w as isolated b y coculturing amoebae in Lake Sabka, a hypersaline lake in Tunisia.Similarly the genus Legionella has been identified in w astew ater, from the Great Salt Lake of Utah, USA, with salinities r anging fr om 3% to 140% (Schwake et al. 2021 ).Salinity also affects the pr e v alence of pisciric kettsiosis, whic h is higher (74%) at salinities > 26 PSU compared to a m uc h lo w er pr e v alence ( < 29%) at salinities < 26 PSU (Bravo et al. 2020 ).In addition to P. salmonis , se v er al species of Piscirickettsiaceae have been isolated from marine, br ac kish, or lake habitats .T his is the case , for example , of F. adeliensis , which is able to adapt to fluctuations in ambient salinity (0%-3.5%)(Vallesi et al. 2019 ).
Another k e y factor that determines the structure of a microbial community is pH.Ho w ever, it is rarely reported: only 0.7% of the samples we investigated contained pH data.Variations in pH have a significant impact on the biological mechanisms of DIGs.For example, C. burnetii , the causative agent of Q fever, replicates in an intr acellular pha gol ysosome at a pH between 4 and 5. Data show that C. burnetii is unable to grow in media with a pH of 6.0 or higher, but cells remain viable (Smith et al. 2019 ).Surprisingly, one study shows that F. tularensis subsp .holarctica grows at a r educed r ate at pH 7.4 compared to pH 6.4, suggesting that pH 7.4 (i.e. the human body) is a challenge to which the bacteria must adapt (Wastella et al. 2018 ).Conv ersel y, ada ptation also occurs when F. tularensis is in a stressful condition with a low pH.Indeed, se v er al c hanges in the pathogen's mac hinery ar e observ ed, suc h as the cessation of maturation of vacuole-containing bacteria, the cessation of fusion with the lysosome and, on the contr ary, a disintegr ation of the v acuole membr ane by mec hanisms not yet elucidated (Klimentova et al. 2019 ).Modification of pH can actually be used as an effective disinfectant in cooling to w ers: conditioning at pH ≥ 9.6 is considered an effective operation to control the pathogenicity of L. pneumophila (Pinel et al. 2021 ).

Impact of molecular mechanisms on the m utualism-par asitism continuum of DIGs
The host immune system is an important barrier against all colonizing micr oor ganisms, fr om those that ar e harmful to those that are beneficial (Belkaid and Hand 2014 ).Mutualistic interactions with micr obes hav e facilitated the radiation of the major eukaryotic lineages , e .g. mitochondria and chloroplasts (Wernegreen 2012 ).In particular, endosymbionts can provide biochemi-cal ca pabilities suc h as photosynthesis, c hemosynthesis, and nitrogen fixation that allow eukaryotes to adapt to new habitats or specialize in specific nutritional niches (Duron et al. 2018a ).Conv ersel y, par asitic inter actions with pathogens hav e been facilitated by the deployment of virulence factors that allow them to sub v ert the host's antimicrobial countermeasures .T he more virulent a pathogen, the greater the degree of damage it can cause in the host, but virulence e v entuall y e volv es to a le v el that optimizes the r epr oduction and tr ansmission r ate of the pathogen (Gomez-Valero and Buchrieser 2019 ).Virulence has evolved through the coevolution of pathogens with their hosts, a major driver of e volution ov er millions of years.Host-pathogen coevolution is widespread in ecosystems, and resulted in the emergence of sophisticated mechanisms for disrupting host functions on one side, and shaping the immune defences of eukaryotic cells on the other side.In this section, we r e vie w two mechanisms that generate different kinds of interactions along the mutualistic-parasitic continuum between endosymbionts and their hosts .T he first is the T4BSS (Fig. 6 ), which is found in most DIGs (all Legionellales , some F rancisellaceae -but not F rancisella -and Piscirickettsia ) and is used by bacteria to inject proteins into the host cytoplasm to resist digestion during phagocytosis (Ghosal et al. 2019 ).The second is the synthesis of biotin (also known as vitamin H or B7), an essential cofactor that is synthesized by a variety of conserved pathways and may be r equir ed for the virulence of certain pathogenic bacteria (Sirithanakorn and Cronan 2021 ).These different systems will be r e vie wed, highlighting their conserv ation status and their role within DIGs.

The Dot/Icm T4BSS
Type IV secretion systems (T4SS) are nanomachines produced by Gr am-negativ e and Gr am-positiv e bacteria to transport macromolecules across their membranes .T he systems are used for a wide range of functions, including the exchange of genetic material between bacterial species, the acquisition of novel genetic material from the external environment, and the delivery of nucleoprotein complexes or effector proteins to recipient cells (Wallden et al. 2010 ).Se v er al T4SS ar e pr esent in DIGs, but one stands out as critical for their interaction with their host: the T4BSS, which tr anslocates pr oteins to the host cytoplasm.It consists of one or se v er al oper ons coding for pr oteins called intr acellular m ultiplication (Icm) or defects in organelle trafficking (Dot) (Vogel et al. 1998, Segal et al. 2005 ).The structur e and function of the pr oteins constituting the T4BSS in L. pneumophila have been extensively revie wed r ecentl y (e.g.Loc kwood et al. 2022 ), and will only be briefly summarized here .T his T4BSS, also r eferr ed to as Dot/Icm system consists of about 27 proteins, including an outer membrane protein (DotH), outer membrane lipoproteins (DotC, DotD, and DotK), a periplasmic protein (IcmX), inner membrane proteins (DotA, DotE, DotF, DotG, DotI, DotJ, DotM, DotP, DotU, DotV, IcmF, IcmT, and IcmV), inner membr ane-associated ATP ases (DotB, DotL, and DotO), and soluble cytosolic proteins (DotN, IcmQ, IcmR, IcmS, and IcmW) (Fig. 6 ) (Ghosal et al. 2019 ).The T4BSS is usually subdivided into a core transmembrane subcomplex (mainly DotC, DotD, DotF, DotG, and DotH) and a coupling protein subcomplex (mainly DotL, DotM, DotN, IcmS, IcmW, and LvgA) (Ghosal et al. 2019 ).T he T4BSS pla ys a k e y role in the interaction of Coxiella  establishment of a replication vacuole and intracellular growth in L. pneumophila and C. burnetii (Kidane et al. 2022 ).Mor e r ecentl y, the discovery of T4BSS homologs in members of the Francisellaceae and Piscirickettsiaceae pushes the origin of T4BSS back to the common ancestor of the orders Legionellales and the two above-mentioned families .For example , a Dot/Icm system has been identified in the fish pathogen P. salmonis , where the phagosome-lysosome fusion e v ent is inhibited during Piscirickettsia infection (Mauel et al. 1999, Fa et al. 2013 ).A homologous T4BSS is also present in F. hongkongensis ( Fastidiosibacteraceae ), possibly acquired by horizontal gene transfer (HGT) from Piscirickettsia (Hugoson et al. 2022 ).
T4BSS is found in all Legionella species studied (except the very reduced insect endosymbiont ' Ca .Legionella polyplacis'), in C. burnetii , the etiological agent of Q fever, in P. salmonis and in Rickettsiella grylli , and is essential for their intracellular growth (Hugoson et al. 2022 ).The functional similarities between the T4BSS of C. burnetii and L. pneumophila are w ell kno wn.For example, the C. burnetii Nine Mile RSA439 genome encodes homologs of 23 of the 26 L. pneumophila Dot/Icm proteins, with amino acid sequence identities r anging fr om 22% to 66% (Larson et al. 2023 ).In the genomes of ' Ca .Berkiella cookevillensis' and ' Ca .Berkiella aquae', 17 and 18 of the 26 Dot/Icm genes share a high degree of homology with those of L. pneumophila (Kidane et al. 2022 ).The situation is similar in P. salmonis, where almost all T4BSS proteins described in L. pneumophila and C. burnetii have been identified in the P. salmonis proteome, including core proteins DotH, DotG, DotF, and DotD (Cortés et al. 2017 ).Ho w e v er, the T4BSS of these four gener a (i.e.Legionella , Coxiella , Rickettsiella , and Piscirickettsia ) show differences (Burstein et al. 2016, Grohmann et al. 2017 ).Indeed, while IcmQ orthologs are found in the Legionella , Coxiella , and Rickettsiella Dot/Icm systems, there is a lack of interspecific complementation by IcmQ for the latter two genera (Segal et al. 2005 ).This is expected, as the L. pneumophila IcmQ protein interacts with the IcmR pr otein, whic h is absent from Coxiella and Rickettsiella , even though functional IcmR homologs have been found in both organisms (Nagai andKubori 2011 , Grohmann et al. 2017 ).
The C. burnetii genome also lacks DotJ, but has a tandem duplication of the gene coding for DotI, while R. grylli has both DotI and DotJ proteins (Segal et al. 2005 , Nagai andKubori 2011 ).DotJ/IcmM and DotI/IcmL genes in L. pneumophila are homologous to each other.DotI and DotJ ar e tightl y bound, inner membr ane integr al pr oteins that ar e essential for Dot/Icm-dependent activities.DotJ consists only of the conserved N-terminal region, whereas DotI has an additional periplasmic domain (Ghosal et al. 2019 ).The similarity between DotJ and DotI suggests that the DotI duplication can substitute for the absence of DotJ, the functional homolog of IcmR (Fig. 6 ) (v an Sc haik et al. 2013 ).This suggests that the DotI gene duplication occurred in a common ancestor of Legionella , Coxiella , and Rickettsiella , and that the DotJ-like protein evolved after species differentiation.
DotV, IcmF, and DotU/IcmH are also absent in Rickettsiella , while IcmF is fr a gmented in Coxiella (v an Sc haik et al. 2013 , Christie et al. 2017 ).The two genes located at the 5' end of the Dot/Icm II region in L. pneumophila ( icmHF ) were found to be 1270 kb a wa y from the rest of the Dot/Icm genes in C. burnetii , and one of these genes ( icmF ) was interrupted by an early stop codon.
A phylogenetic study of Rickettsiella popilliae and R. grylli sho w ed that these bacteria carry genes orthologous to dotB and dotO (Nagai and Kubori 2011 ).An interspecific complementation study of L. pneumophila Dot/Icm mutants with homologous C. burnetii Dot/Icm proteins was performed.This analysis identified four C. burnetii Dot/Icm proteins that can be expected to replace their L. pneumophila homologues (IcmS, IcmW, IcmT, and DotB) and six other proteins (IcmQ, IcmP/DotM, IcmO/DotL, IcmJ/DotN, IcmB/DotO, and IcmX) that cannot (Zamboni et al. 2003, Segal et al. 2005, van Schaik et al. 2013 ).This functional substitution of their L. pneumophila counterparts in intracellular replication sho w ed that the Coxiella Dot/Icm system is functional and plays an essential role in interactions with its host cells (Zamboni et al. 2003, Zusman et al. 2003 ).
Differ ential expr ession of dotH and dotG was observed in P. salmonis , whic h may corr elate with the establishment of the infectious process when bacteria inject virulence factors into host cells .T he secretion of effector proteins by the T4BSS in P. salmonis could pr omote l ysosomal esca pe of bacteria and favour intracellular survi val.Unlik e bacteria such as Legionella or Coxiella , which hav e m ultiple div er gent copies of T4BSS genes, r eflecting functional diversity , P .salmonis , shows div er gence in the expr ession le vels of T4BSS genes rather than in copy number (Fig. 6 ) (Nourdin-Galindo et al. 2017 ).
Studies have shown that the T4BSS of the genus Legionella harbours three Legionella-specific proteins, IcmX, DotJ, and DotF.The first is subject to high r ecombination r ates (Gomez-Valer o and Buchrieser 2019 ), located on the surface of the bacteria, and therefor e likel y to be exposed to the host.The second, DotJ, is located in the inner membrane and is a distant paralog of DotI, with which it forms a heteroduplex (Kuroda et al. 2015 ).The third, DotF, has a periplasmic portion that would be under positive selection and would be involved in substrate recognition (Sutherland et al. 2013 ).
Even if the Dot/Icm system is part of the core Legionella genome, some differences in the organization of the constituent proteins ar e observ ed between the different Legionella species, e v en if it r emains highl y conserv ed (Christie et al. 2017 , Gomez-Valer o andBuc hrieser 2019 ).Indeed, these differ ences ar e due to gene insertions of variable sizes, for example, a se v en-gene insertion between icmB and icmF in L. brunensi , compared to a single gene between the same two genes in L. pneumophila , and no intermediate genes in L. quinlivanii (Burstein et al. 2016 ).While these inserted genes have not been definiti vely link ed to the Dot/Icm secretion system, the possibility that they might play a role in this system, potentiall y acquir ed thr ough HGT, should not be discounted.

Di v ersity and functions of the effectors
Intr acellular gr owth of DIGs r equir es the T4BSS, encoded by the d ot / icm genes (see section abov e), whic h tr anslocates bacterial proteins into the host cytoplasm, where they manipulate various host cellular processes (O'Connor et al. 2011 ).These proteins, called effectors , pla y a major role in DIG virulence and are welldescribed in L. pneumophila , the causativ e a gent of Legionnaires' disease .T he function of these effectors has also been r ecentl y r e vie wed (e.g.Loc kwood et al. 2022 , Chauhan et al. 2023 ).Effectors modify the host behaviour in multiple ways, but their effects can be br oadl y divided in two categories .T he first is to avoid being killed by the host, among others by pr e v enting the fusion of the phagosome with the lysosome, by modulating signalling pathwa ys in volved in pathogen recognition, or by preventing apoptosis.The second is to favour the exploitation of the host's resources by manipulating the endosome tr affic king system and hijacking the ubiquitin pathway.
In Legionella , effectors make up about 10% of the genome (Grohmann et al. 2017, Hilbi et al. 2017 ).Studies show that the pangenome of the genus Legionella contains over 1600 families (18 000 genes) of protein effectors, but that only eight are conserv ed acr oss the genus (Burstein et al. 2016, Hilbi et al. 2017, Gomez-Valer o et al. 2019 ).Onl y a subset of these effectors have been c har acterized, and in man y cases m utations of the effector genes do not lead to discernible phenotypes, pr obabl y due to functional redundancy.To date, some of the effectors have been shown to play a role in modifying host cellular processes to establish a connection between the bacteria and the host cell, allowing L. pneumophila to colonize the host cell (Kubori and Nagai 2016 ).While the T4BSS is highly conserved, most effectors are dispensable when deleted individuall y.Indeed, sim ultaneous deletion of multiple substrates had little effect on mouse macr opha ge growth (O'Connor et al. 2011 ).Howe v er, the r epertoir e of effectors v aries consider abl y between these species.An anal ysis of putative T4BSS effectors from Legionella longbeachae , the second most common cause of Legionnaires' disease, revealed that only about 50% of the virulence factors described in L. pneumophila were also present in the L. longbeachae genome (Gomez-Valero et al. 2019 ).One study found that 258 effectors (42%) were species-specific , i.e .observ ed in onl y one of the 50 Legionella species analysed (Burstein et al. 2016 ).Excluding L. pneumophila , the species with the highest number of unique putative effectors is L. waltersii , with 23 specific effectors.In addition, L. pneumophila encodes a unique family of translocated effectors called meta-effectors, which are effectors that interact with each other and regulate other L. pneumophila effectors in host cells (Urbanus et al. 2016 ).A genetic screen identified 20 possible interactions between effectors.Howe v er, for most meta-effectors, no direct interactions have been experimentall y demonstr ated.Curr entl y, 10% of L. pneumophila effectors have been shown to be part of the effector protein interactome, suggesting a str ong pr esence of meta-effectors (Grohmann et al. 2017 ).One study has r ecentl y described the meta-effector, MesI, whic h pr omotes L. pneumophila virulence by regulating the cytotoxic effector SidI.When MesI and SidI are uncoupled, SidI is toxic to L. pneumophila ( in vitro ) and induces se v er e bacterial degradation in host cells .Furthermore , MesI translocation appeared to be essential for intracellular replication, demonstrating that intr abacterial r egulation of SidI contributes to L. pneumophila virulence (van Schaik et al. 2013, Chauhan et al. 2023 ).Meta-effectors are a k e y element of L. pneumophila virulence, as se v er al ar e r equir ed for intr acellular r eplication.Recentl y, par a-effectors (fr om the Greek para meaning besides but also contrary to ), a new category of effectors has been introduced (Schator et al. 2023 ).Some effectors function as pairs of highly interdependent effectors that finetune host cell gene expression and promote bacterial intracellular replication.This has been demonstrated for two chromatin modifying effectors (RomA and LphD) in L. pneumophila .Using mutation and virulence assa ys , it w as sho wn that the presence of only one of these two effectors impairs intracellular replication, whereas a double knock-out ( lphD romA ) can restore intracellular replication (Schator et al. 2023 ).
The effectors that pass through the T4BSS of C. burnetii have not been the subject of such in-depth studies, largely due to the difficulty to grow Coxiella axenically (Beare et al. 2009 ).To date, 150 pr otein substr ates hav e been identified in C. burnetii , r epr esenting ∼6% of the open reading frames in its genome (Chen et al. 2010, Carey et al. 2011, Qiu and Luo 2017 ).Only a few C. burnetii effector pr oteins hav e been functionall y c har acterized, with r oles in interfering with vesicular trafficking, host transcription, and apoptosis (Larson et al. 2016, 2019, Lührmann et al. 2017 ).In some cases, the cellular targets of the effectors have been identified.For example, AnkG, CaeA, and CaeB can inhibit host cell a poptosis.Mor e specifically, CaeA localizes to the nuclei of infected cells where it activates the expression of surviving, an inhibitor of activated caspases, which can contribute to the induction of apoptosis (van Schaik et al. 2013 , Qiu andLuo 2017 ).Other effectors are specificall y involv ed in lipid metabolism of the Coxiella -containing vacuole, host gene expr ession, autopha gy, cell death, and imm unity (Qiu andLuo 2017 , Larson et al. 2023 ).Only six C. burnetii effectors have homologs in L. pneumophila , and in contrast to the extensiv e functional r edundanc y betw een L. pneumophila effectors, C. burnetii mutants lacking a single effector often lose the ability to gr ow optimall y in host cells (Hilbi et al. 2017, Lührmann et al. 2017 ).The a ppar ent lac k of r edundanc y betw een effectors in this species may be due to the compar ativ el y narr ow host r ange of C. burnetii compared to L. pneumophila , suggesting their important role in virulence and host ada ptation.(Dur on et al. 2018a, Santos-Garcia et al . 2023 ).
Only four effectors have been identified by genetic screening in P. salmonis , all homologous to L. pneumophila and C. burnetii effectors (Grohmann et al. 2017 ).The four proteins have a typical Dot/Icm secretion signal at the C-terminus and are overexpressed during in vitro infections, suggesting a potential involvement in intr acellular surviv al and pr olifer ation of P. salmonis (Labr a et al. 2016 ).Furthermore, these four effectors all have eukaryotic-like pr otein domains, r e v ealing a possible common function during translocation to the eukaryotic host during the establishment of pathogen-host relationships.
Effectors may also play a role in environmental adaptation, as suggested by an analysis that r e v ealed 51 ne w putativ e effectors specific to L. longbeac hae , whic h may be related to its different lifestyle compared to L. pneumophila (Gomez-Valero et al. 2019 ).Indeed, L. longbeachae is found in wet soils and loams, and genes possibl y originating fr om plants hav e been identified, suc h as pr oteins with pentatricopeptide repeat domains, a significantly protein family in plants.A subpopulation of effectors has also been correlated with host-specific replication of L. pneumophila in A. castellanii, A. polyphaga, H. veriformis , and N. gruberi (Chauhan and Shames 2021 ).The central effector MavN, which plays a role in envir onmental ada ptation, has also been identified in L. pneumophila and R. grylli (Burstein et al. 2016 ).MavN was r ecentl y found to be an iron transporter (Christenson et al. 2019 ).
The evolution of multiple mechanisms to adapt to different environmental conditions and subvert the eukaryotic host is likely due to the molecular arms race that has de v eloped between differ ent DIG or ganisms and the br oad spectrum of pr otozoan hosts encountered in their natural environment (Grohmann et al. 2017 ).This suggests that many effectors with potentially important functions in virulence hav e r ecentl y been acquired by HGT, highlighting DIGs as a highly dynamic system capable of rapid adaptation (Burstein et al. 2016(Burstein et al. , Říhová et al. 2017 ) ).

Eukaryotic domains
Effector proteins often carry conserved eukaryotic protein domains.A eukaryotic domain has been defined as a protein domain found in > 75% of eukaryotic genomes and < 25% of prokaryotic genomes (Mondino et al. 2020 ).These eukaryotic protein domains ar e v ery important in host-micr oor ganism inter actions: once tr ansferr ed into the micr oor ganism genome, their original function that was in the host is altered to the benefit of the micr oor ganism.Indeed, the majority of these genes are likely to have been acquired by HGT directly from protozoa, highlighting the importance and coevolution of DIG interactions with their host (Chauhan and Shames 2021 ) The most abundant eukaryotic domains identified in DIGs ar e ankyrin r epeats (Gomez-Valer o and Buc hrieser 2019 ).Indeed, more than 20 genes encoding proteins containing eukaryotic ankyrin repeats have been identified in Coxiella .Ankyrin repeats ar e pr otein-pr otein inter action motifs of tandeml y r epeated modules of 30-34 amino acids found in eukaryotic proteins in-volv ed in v arious cellular functions, including tr anscriptional r egulation, signal transduction, vesicular trafficking, and cytoskeletal integrity (Kidane et al. 2022 ).A large majority of the effectors identified in the genomes of P. salmonis , Legionella santicrucis , and Legionella massiliensis harbour ankyrin repeats, with the latter two genomes encoding 41 and 39 ankyrin domains, respectiv el y.Ankyrin motifs are frequently associated with other eukaryotic motifs and are associated with eukaryotic F-bo x, U-bo x, Rab, or SET domains, suggesting that manipulation of the host ubiquitin system is a fundamental virulence strategy (Labra et al. 2016, Grohmann et al. 2017, Qiu and Luo 2017, Gomez-Valero et al. 2019, Chauhan and Shames 2021, Kidane et al. 2022 ).F-boxlike domains are also well described in L. pneumophila and C. burnetii .Based on their functions in eukaryotic cells, this group of proteins pr esumabl y pr omotes pr oteasome-mediated pr otein degr adation (van Schaik et al. 2013 ).For example, AnkB (F-box protein) in L. pneumophila targets nonessential host proteins (via ubiquitylation) for degradation by the 26S proteasome, thereby providing an energy source for its growth.L. pneumophila also mimics eukary otic RN As, such as miRN As, to interfere with eukary otic regulatory mechanisms .For example , L. pneumophila uses extracellular vesicles to translocate small bacterial RNAs (e.g.RsmY and tRNA-Phe) into host cells to interfere with host defence pathways (i.e.downr egulating the expr ession of k e y sensors and regulators of the host cell innate immune response) (Sahr et al. 2022 ).

Biotin
Biotin synthesis is also essential for the virulence of some human pathogens, such as F. tularensis and some Coxiellaceae (Santos et al. 2003, Feng et al. 2014 ).Biotin (vitamin B7 or H) is essential in all aspects of life as it plays a k e y role in central metabolic processes involving carbo xylation, decarbo xylation, and transcarbo xylation (Sirithanak orn and Cr onan 2021 ).The function of biotin synthesis is well described for F rancisella .A F rancisella protein, FTN_0818, was identified as involved in biotin biosynthesis and at the same time essential for intracellular replication.It is required for rapid esca pe fr om the Francisella -containing pha gosome, suggesting that biotin may be a limiting factor: when absent, it confines cytosolic pathogens to the pha gosome, bloc king their escape and preventing them from reaching their replication niche in the cytoplasm.Biotin sequestration may be a form of nutritional immunity by the host's innate immune system and supports the idea that biotin may be a critical and limited resource during infection and is ther efor e corr elated with Francisella virulence (Napier et al. 2012, Feng et al. 2014, Duron et al. 2018b ).The role of biotin synthesis in DIG virulence has also been highlighted in C. burnetii .Indeed, blocking biotin biosynthesis inhibits the growth of C. burnetii on specific axenic media, suggesting that biotin is r equir ed for the growth and development of this pathogen (Moses et al. 2017, Duron et al. 2018b, Santos-Garcia et al . 2023 ).Ho w e v er, the bioc hemical/metabolic mec hanism used by some DIGs to synthesize biotin and thus facilitate their infectivity remains unknown.On the other end of the spectrum, the acquisition-through HGT-of a biotin synthesis operon contributed to shifting the position of a Legionella species along the par asitism-m utualism continuum.Indeed, in contrast to other Legionella species, which are facultative intracellular pathogens of various protists, ' Ca.Legionella polyplacis', is an obligate mutualistic endosymbiont of tic ks, pr esumabl y pr o viding its host with biotin ( Řího vá et al. 2017 ).
In summary, the diverse repertoire of effectors in different DIG species reflects a high degree of evolutionary plasticity, with constant acquisitions , losses , and adaptation of genetic material from host cells or coinfecting pathogens supporting DIG virulence.Ho w e v er, the inability to identify effectors remains a major challenge in the study of host-DIG interactions along the mutualismpar asitism continuum.Furthermor e, a better c har acterization of effectors will allow us to know whether the effector r epertoir e of a particular DIG strain reflects the ecosystem from which it was originally isolated, and whether the hosts of that particular niche have an impact on the composition and diversity of effectors.

Influence of the micr obiota-inter action with other bacteria and endosymbionts
Most ecosystems are populated by large numbers of diverse micr oor ganisms that interact with each other to form complex networks of ecological interactions .T he possible combination of positi ve ( + ), negati ve ( −), or neutral (0) outcomes for both partners in the interaction makes it possible to classify different types of interactions along the mutualism-parasitism continuum, for example mutualism ( + ,+ ), commensalism (0,+ or + ,0), amensalism (0,− or −,0), parasitism ( + ,− or −,+ ), and competition ( −,−) (Lidicker 1979 , Faust andRaes 2012 ).Some of these micr oor ganisms can colonize the surface and/or internal parts of other organisms such as plants and animals, adding a further level of complexity to the interactions.In addition, abiotic factors (e.g.physical and c hemical par ameters) also sha pe the natur e and complexity of microbial interactions (Paniagua et al. 2020 ).These microbial interactions can be both intraspecific and interspecific, ranging from simple short-term interactions to complex long-term interactions (Moënne-Loccoz et al. 2014 ).
All DIGs are present in a wide range of ecosystems (see abo ve , section -Environmental distribution of DIGs) and interact along the m utualism-par asitism continuum with v arious micr oor ganisms present in the community.Due to their distribution and lifestyle (i.e.free-living and intracellular), the nature and underlying mechanisms of these interactions are often unknown and difficult to study within ecosystems.In this section, we will r e vie w the interactions between DIGs and different microorganisms in thr ee differ ent contexts: inter actions outside the host, intr acellular interactions, and interactions within multicellular hosts.

Interactions outside the host
Despite their mainly intracellular lifestyle, DIGs have been observ ed to inter act with differ ent micr oor ganisms in biofilms when they are outside of their host.Biofilms r epr esent important environments for the ecology of se v er al DIG species, as they provide se v er al functions, suc h as pr otection fr om biocides, a source of nutrition, and a source of dissemination (Stewart 1993, Kim et al. 2002, Temmerman et al. 2006, Hsu et al. 2011 ).Mor eov er, as biofilms contain the bulk of microbial biomass, they attract micr obial gr azers and ar e wher e most biological inter actions occur between microbial species (Flemming et al. 2002 , Lau andAshbolt 2009 ).
Pr otozoa ar e micr obial gr azers that play a significant r ole in shaping and structuring the bacterial community of an ecosystem, particularly in biofilms: they impact the microbial loop, carbon flux, and nutrient cycling of biofilms (Hahn andHöfle 2001 , Bonkowski 2004 ).DIGs that infect and kill protozoa will reduce the le v el of gr azing and r educe the pr essur e on the heter otr ophic and primary producer populations in an ecosystem.This may explain the establishment of positive interactions between DIGs and other micr oor ganisms in the comm unity.For instance, Flavobacterium breve , Brevundimonas sp., and Fischerella sp.(c y anobacteria isolated from microbial mats), have been observed to nutritionally supplement L. pneumophila strains (Tison et al. 1980, Wado wsk y and Yee 1983, P ar anja pe et al . 2020a ).Though the exact nature and reason for this supplementation are unknown, it is thought to allow increased numbers of L. pneumophila around the supplier species .T his in turn increases the probability that L. pneumophila cells are ingested by grazers and decreases the chances of the supplier species being ingested (P ar anja pe et al .2020b ).An added benefit is that grazers may e v entuall y die if they are sensitive to L. pneumophila .Conv ersel y, other species in the biofilm community can negativ el y inter act (i.e.amensalism, par asitism, and competition) with some DIG species .T hus , species in a biofilm can produce antimicrobial molecules, which can either inhibit or kill DIG species .For instance , Bacillus , Pseudomonas , Stenotrophomonas , Chryseobacterium , Staphylococcus , Cupriavidus , Aeromonas , and Flavobacterium can inhibit L. pneumophila through the production of various antimicr obials (e.g.pr oteases and surfactin), leading to the establishment of amensalism interactions (Loiseau et al. 2015, Faucher et al. 2022, P ar anja pe et al. 2022 ).As DIGs contain se v er al human pathogens, identifying and isolating species that can produce antimicr obials effectiv e a gainst these pathogens may pr ov e v aluable for public health issues.
Some eukaryotic and bacterial species in biofilms can also physicall y dama ge or destr oy DIGs, and ar e usuall y pr edators or parasites .For instance , the eukaryotic heterolobosean amoeba Solumistris palustris and se v er al cercozoan mor photypes consume L. pneumophila cells for nutrition (Anderson et al. 2011 ); in contrast, L. steelei is able to destroy S. palustris through a food poisoning-like method, indicating a DIG's specificity in the pr edator-pr ey r elation (Amaro et al. 2015 ).Another example of predatory interaction is the bacterial species Bdellovibrio bacteriovorus , which has been shown to prey on several species of Legionella and Francisella (Tomov et al. 1982, Russo et al. 2015 ).Finall y, bacteriopha ges can be consider ed as anta gonists, but r esearc h on pha ge-DIG r elationships is still limited and contr ov ersial (see L. pneumophila phage in Cav allar o et al. 2022 ).One potential hurdle for the study of these phages may be the requirement of a tripartite model involving a phage, a DIG species, and an a ppr opriate host species.Nonetheless, se v er al vir al genetic elements hav e been discov er ed in v arious DIG species, suc h as complete pr opha ge sequences in Legionella micdadei , Cysteiniphilum littorale ( Fastidiosibacteraceae ), and Francisella hispaniensis , and CRISPR-Cas systems in L. pneumophila and F. novicida (Schunder et al. 2013, Gomez-Valero et al. 2014, Tla pák et al. 2018, Deec ker et al. 2021, Qian et al. 2023 ).Furthermor e, vir al particles have been observed and imaged to infect L. pneumophila and P. salmonis , and r ecentl y UV r adiation w as sho wn to induce the assembly of noninfectious viral particles in F. hispaniensis (Yuksel et al. 2001, Lammertyn et al. 2008, Köppen et al. 2021 ).All in all, these results suggest that phages do exist and inter act negativ el y with DIGs in natur e, but mor e r esearc h is needed to confirm this hypothesis.
Indir ect inter actions between DIGs and se v er al species also take place in the biofilm environment.An indirect biological interaction can be defined as the influence that an organism has on another through its interaction with another organism (Moon et al. 2010 ).A classic example is the trophic cascade effect where predation of one organism either positiv el y or negativ el y affects another organism (Menge 1995 ).The parasitism of protozoan grazers by DIGs, such as L. pneumophila already mentioned abo ve , illustrates the indirect effect of a trophic cascade on the grazer's prey population.The interactions between L. pneumophila , amoebae and nematodes have been documented in biofilms and are examples of trophic cascade (Brassinga et al. 2010, Rasch et al. 2016 ).For example, the nematode Plectus similis feeds on various bacteria and amoebae, such as A. castellanii.The nematode, when pr esented with fr ee-living L. pneumophila , displays r educed feeding behaviour than when presented with Esc heric hia coli , suggesting that it activ el y avoids ingesting the former.(Hemmerling et al. 2023 ).Ho w e v er, the nematode will feed on A. castellanii infected with L. pneumophila at similar rates than noninfected A. castellanii , allowing colonization of the gut of the nematode by L. pneumophila , which would not happen if the bacteria was free living (Hemmerling et al. 2023 ).The impact of the colonization of L. pneumophila on the gut of the nematode is not well-understood yet, but there is some emerging evidence that L. pneumophila negatively impacts the nematode C. elegans (Rasch et al. 2016 ), which is corroborated by the fact that free-living L. pneumophila are not preferred by P. similis .
Pr obiotic a ppr oac hes hav e garner ed inter est as a potential method to reduce or eliminate pathogens, such as L. pneumophila , fr om v arious r ele v ant ecosystems, including human gut or engineered water systems (Wang et al. 2013, Veiga et al. 2020, Cavallaro et al. 2022 ).Ho w ever, with these approaches, indirect biological inter actions hav e to be taken into account.Indeed, the purposeful elimination from an ecosystem of L. pneumophila , known to dominate water systems over other Legionella species, for example by adding the predator S. palustris , could increase the abundance of non-pneumophila Legionella species, which might in turn also be pathogens for humans (Wéry et al. 2008, Amaro et al. 2015, Salinas et al. 2021 ).T hus , the study of the indir ect biological inter actions of the members of the microbiome will undoubtedly be useful to pr edict, v erify, model, and assess control strategies of pathogens.

Intr acellular inter actions
Man y DIG species ar e associated with envir onmental unicellular protozoans, and the biological interactions that occur between these two actors are numerous and complex.Protozoan hosts are a source of nutrition for DIG species that are colonized by them.Nutrition is usuall y acquir ed thr ough par asitic inter actions, with species within the Legionella genus being a prime example (Isberg et al. 2009 , Gomez-Valero andBuchrieser 2019 ).Mutualistic nutritional interactions have yet to be observed between DIGs and protozoa; ho w ever, novel species of DIGs reside in amoebas without causing lysis.' Ca .Rhogoubacter', ' Ca .Pokemonas', ' Ca .Fiscibacter', ' Ca .Occultobacter', and ' Ca .Nucleophilum' are all candidate genera closely related to the Coxiella and Legionella genera (Schulz et al. 2015, Pohl et al. 2021, Solbach et al. 2021 ).In these or ganisms, the lac k of l ysis of the host upon infection suggests a mutualistic relation between the partners, potentially based on nutritional supplementation.Mor eov er, a r ecentl y discov er ed and v ery r educed endosymbiont (' Ca.Azoamicus ciliaticola') displays a clear mutualistic relationship with its host, an anaerobic, amitoc hondriate ciliate, wher e the mitoc hondrial ener getic functions have been replaced by the DIG endosymbiont (Graf et al. 2021 ).
Protozoan host species are also a source of protection for DIG species.Se v er al pr otozoan species ar e kno wn to enc yst, providing pr otection fr om envir onmental str esses for bacteria-including DIGs, in particular Legionella spp .-present within them (Lambr ec ht et al. 2015 ).As bacteria can avoid detection when localized in cysts, this phenomenon has qualified protozoa hosts as 'Trojan horses' (Barker and Brown 1994 ).In a different example of protection, Caedibacter taeniospir alis ( Fastidiosibacter aceae ), a mutualistic endosymbiont of Paramecium species, is capable of conferring a killer trait to its host by the production of R-bodies, constituted of insoluble protein ribbons (Heruth et al. 1994, Beier et al. 2002 ).
Paramecium cells that do not harbour the endosymbiont and ingest the R-bodies die from a toxin-related mechanism (Schrallhammer et al. 2018 ).The Paramecium -containing endosymbionts thus gain an adv anta ge for resources and protection by destroying their competitors.Another function of protozoan hosts, and especially free-living amoebae, that has been hypothesized is that they serve as 'tr aining gr ounds' for the de v elopment of intr acellular bacterial human pathogens (Molmeret et al. 2005 ).This hypothesis is based on the fact that gr azing pr otozoa place selection pr essur e on their prey and generate resistance to grazing (Amaro et al. 2015 ).This resistance can then be applied to human immune cells, such as macr opha ges, that use phagocytosis to kill pathogens.For example, the Legionella and Francisella genera contain species that are pathogenic to humans and acquired their virulence factors from their coevolution with amoebae (Barker and Brown 1994 ).Interactions between protozoa and DIGs can lead to the emergence of se v er al DIG species with negative consequences for humans.
As well as interacting directly with their unicellular host, DIGs can also interact with its microbiota.In fact, unicellular protozoa can contain small micr obiota, r anging fr om one m utualistic endosymbiont to se v er al dozen gener a of differ ent bacteria (Delafont et al. 2013, Tsao et al. 2017, Solbach et al. 2021, Delumeau et al. 2023 ).Some social amoebae, such as Dictyostelium discoideum , can 'farm' their microbiome for their own benefit (Br oc k et al. 2011 ).One function for this microbiota is to defend the host against parasitic DIGs of amoeba.For example, Acanthamoeba S13WT harbours the chlamydial endosymbionts Protoc hlam ydia amoebophila and Neoc hlam ydia sp.These two endosymbionts pr otect their host against infections of L. pneumophila (Maita et al. 2018, König et al. 2019 ).The pr otection mec hanisms ar e not full y understood, and it has been suggested that they are different for the two endosymbionts.For P. amoebophila , the endosymbiont changes the expr ession pr ofile of the infecting L. pneumophila so that it cannot pr ogr ess to its tr ansmissiv e phase, making it impossible to infect subsequent amoeba hosts in the culture (König et al. 2019 ).In contr ast, Neoc hlam ydia is suggested to pr e v ent L. pneumophila from entering the host (Maita et al. 2018 ).Moreover, coinfection studies of macr opha ges suggest that DIGs can either hav e neutr al or beneficial interactions with other microbes in the intracellular medium.Indeed, coinfection of C. burnetii and L. pneumophila in v arious imm une cells sho w ed that both DIGs were compartmentalized in different vacuoles (Sauer et al. 2005 ).This compartmentalization a ppear ed to separ ate both DIG species and limit competition, creating a neutral effect on the two pathogens' ability to grow inside the immune cells .Moreo ver, coinfection between L. pneumophila and Brucella neotomae in macr opha ges suggested that L. pneumophila promotes the growth of the latter bacterial species (Kang and Kirby 2017 ).This interaction was parasitical as B. neotomae reduced the fitness of L. pneumophila (Kang and Kirby 2017 ).In another example, different species of amoebae infected with L. pneumophila were found to ingest fewer E. coli cells (Shaheen and Ashbolt 2021 ).

Interaction inside multicellular hosts
A large number of DIG species form relationships with arthropods .T hey can be found in salivary glands, in the midgut, or malpighian tubules, (Braendle et al. 2003, Kl yac hk o et al. 2007, Duron et al. 2020 ); in contrast to other symbiont groups, DIGs are often not located in specialized organs or cells.Some DIG species have established mutualistic interactions with arthropods, such as Coxiella -like endosymbionts present in oocytes in tic ks (thr ough v ertical tr ansmission) (Baumann 2005, Kl yac hk o et al. 2007 ) and Francisella -like endosymbionts, which dominate in the microbiome of American dog ticks (Travanty et al. 2019 ).The most widely described mutualistic interaction is through vitamin B supplementation provided by the endosymbiont to its host.For instance, a Coxiella -like endosymbiont of Lone star ticks provides vitamin thiamine (B1), riboflavin (B2), niacin (B3), pantothenic acid (B5), pyridoxine (B6), and folate (B9) to its host; similarly, a Francisella -like endosymbiont of hard ticks harbours the complete pathway for vitamins B2, B7 (biotin), and B9 (Smith et al. 2015, Gerhart et al. 2018 ).Vitamin B supplementation seems to be a conv er gent ada ptation to the nutritional needs of the bloodsucking host as blood meals are deficient in vitamin B (Duron et al. 2018b ).Ther efor e, this supplementation by mutualistic DIGs can be observed for various bloodsucking arthropods, such as lice and ticks (Smith et al. 2015, Říhová et al. 2017, Duron et al. 2018b ).Genes related to vitamin B metabolism have been acquired horizontally by these DIGs, suggesting a r elativ el y r ecent endosymbiosis e v ent (Smith et al. 2015(Smith et al. , Říhová et al. 2017 ) ).To date, vitamin B supplementation is the only known mutualistic interaction between arthropods and DIGs, probably because most of the research has been focused on bloodsucking arthropods.
Though vitamin supplementation seems to be the most pr e v alent interaction between DIGs and arthropods, the dynamics of these interactions are quite diverse.Indeed, certain symbioses seem to occur in a duality with two species of bacteria that complement each other's metabolic pathway for the nutrient required for the host.This has been observed in the Hyalomma marginatum tic k, wher e a Francisella -like endosymbiont supplements the host with folate and ribofla vin.T his endosymbiont, ho w e v er, lac ks the capacity to produce biotin, due to extensive genome degradation (Buysse et al. 2021 ).Compensation for biotin occurs through Midic hloria species, whic h hav e intact genes for biotin synthesis (Buysse et al. 2021 ).In contrast, certain DIGs such as Coxiella -like, F rancisella -like , and Rickettsiella ha ve established competition-type interaction in various tick species.Several of these species appear to offer the same type of nutritional supplementation interaction with their host.In this case, there is a competition between the various species for establishment of an exclusive mutualistic relation with their host, as a host gains no advantage by carrying many endosymbionts with identical functions (Kumar et al. 2022 ).As a r esult, ther e seems to be replacements, and even extinction, of se v er al Coxiella -lik e symbionts by Francisella -lik e symbionts in se v er al linea ges of tic ks (Buysse et al. 2022, Kumar et al 2022 ).Mor e pr ecisel y, in a study by Duron et al. ( 2017 ) Coxiella -like symbionts were found at a prevalence level of 0.68 in ticks with a single endosymbiont species (ticks can be infected by multiple endosymbionts).Ho w e v er, the pr e v alence of Coxiella -like symbionts was reduced to zero and around by half in ticks that had either Rickettsiella -like or Francisella -like symbionts as secondary endosymbionts, r espectiv el y (Dur on et al. 2017 ).Another interaction has been observed between Rickettsiella and Hamiltonella , a facultativ e symbiont, r esulting in a negative effect on European populations of the pea aphid Acyrthosiphon pisum .Infection with Rickettsiella alone had virtually only neutral effects on host fitness, while Rickettsiella-Hamiltonella coinfection had negative effects (Tsuchida et al. 2014 ).
The interactions between other multicellular organisms (other than arthropods) and DIG species are less described in the liter atur e. Ho w e v er, most of these described interactions seem to be pathogenic.For instance, Legionella spp., F. tularensis , C. burnetii , and P. salmonis are all known to cause disease in various mammals and fish species.In these cases, direct and indirect interactions between the various immune systems, tissue cells, and the pathogens occur.For example, Piscirickettsia was detected in the digesta and intestinal mucosa of Atlantic salmon.Analysis of the microbiota identified a list of 27 families (e.g.Lactobacillaceae , Flavobacteriaceae , and Tenacibaculum ) with negative associations with Piscirickettsiaceae in infected fish, and another set of 10 families with positive associations (Coca et al. 2023 ).This network analysis suggests that there may be interactions between these bacteria that may produce a metabolic phenotype that favours the virulence of P. salmonis , although other factors, such as the age of the host, the impact of other bacteria on the immune system, or certain physico-c hemical par ameters in the environment of the host might also create those patterns (Coca et al. 2023 ).In addition, P. salmonis and F. noatunensis subsp .noatunensis were detected at the same time in the gut of Salmo salar , suggesting that the interactions between DIGs also occur in other hosts than arthropods (Karlsen et al. 2017 ).Mor eov er, DIGs ar e also being r ecover ed fr om less well-established and studied animals.For example, Legionellaceae and Coxiellaceae sequences have been r ecov er ed from marine sponges (Yang et al. 2022 ).Furthermore , noncon ventional animal models, such as zebrafish, nematodes, and insect larvae ( Galleria mellonella ), are also being used to study infections of L. pneumophila (Leseigneur and Buchrieser 2023 ).These discoveries point out that DIGs may be associated with a wider range of animals than pr e viousl y thought, and that certain species may be able to adapt to novel animal species.Ho w ever, most of these pathogenic inter actions hav e been compr ehensiv el y r e vie wed r ecently and they will not be discussed in this section (see Liu andShin 2019 , Rozas-Serri 2022 ).
In summary, DIGs are capable of establishing numerous interactions along the m utualism-par asitism continuum within various en vironments .Ho w ever, kno wledge of the nature of these interactions is limited, in particular because most research focuses solel y on arthr opods and/or on DIGs of public health concern (e.g.L. pneumophila , C. burnetii , and so on).Furthermore, as the geogr a phical distribution of DIGs is fairly wide (see first section), DIGs associated with unicellular and/or multicellular hosts could differ between certain geogr a phical ar eas (i.e.tr opical r ainfor ests v ersus more arid regions).Differences in the structure and composition of the bacterial community of certain arthropods and mammals between habitats and geogr a phical sites have already been documented (Namina et al. 2023 ).These results could ther efor e indicate the existence of specific interactions between endosymbionts, hosts and the surr ounding envir onment within different ecological niches.

Concluding remarks
This r e vie w highlights links between envir onmental, e volutionary, and molecular mechanisms of ancient host-endosymbiont interactions along the mutualism-parasitism continuum.The evolution of host-endosymbiont interactions in relation to current envir onmental tr ends is important, for example, to predict the potential increase in vector-borne diseases associated with envir onmental c hanges .T he en vironmental distribution of DIGs is global, and environmental factors influence their life cycle and virulence.As the global temper atur e is expected to increase by about 1.5 • C by 2050, it is important to take these environmental factors into account.Future work should, therefore, closely examine the influence of environmental factors and the microbiota on the pathogenicity of DIGs.Given the broad global distribution and ada ptiv e ca pacity of DIG endosymbionts, ther e is a significant risk of novel pathogens emerging in the coming decades.Specific molecular mechanisms (T4BSS and biotin biosynthesis) help to facilitate symbiotic interactions across the m utualism-par asitism continuum with specific functions (facilitation of cell adhesion, injection of effectors and virulence factors during host infection, excretion of toxic compounds, and so on).The T4BSS is used by bacteria to inject effector proteins into the host cytoplasm, and effector proteins can be acquir ed fr om hosts thr ough HGT.These mec hanisms gr eatl y facilitate quic k shifts in symbiotic interactions along the parasite-mutualism continuum.In addition, endosymbionts interact not only with their hosts, but also with the micr oor ganisms pr esent in their environment.Indeed, the host microbiota may influence host-bacterial interactions, such as the interactions between Coxiella and Francisella in ticks .T his review highlights the importance of the microbiota in symbiotic inter actions acr oss the m utualism-par asitism continuum.
Among the DIG species discussed abo ve , the majority of examples comes from the study of current human ( Legionella , Coxiella , and Francisella ) or fish ( Piscirickettsia ) pathogens, for obvious reasons.Ho w ever, to better understand the emergence of these pathogens, other groups should also be the subject of more researc h.Among others, the v ery div erse gr oup r elated to Aquicella (Fig. 1 ) (Santos et al. 2003 ), or the un usual intran uclear endosymbiont ' Ca.Berkiella' (Kidane et al. 2022 ) are worth studying further.
In conclusion, there is a need for further multidisciplinary research into the evolution of the molecular mechanisms and interactions of endosymbionts with the host microbiota to understand their pathogenic potential.This will allow us to identify the environmental indices and characteristics and community signatures that influence the pathogenicity of these endosymbionts, while de v eloping public health pr e v ention and control strategies.

Figure 1 .
Figure 1.Simplified phylogeny of DIGs.The tree is based on the bac120 tree obtained from GTDB (Parks et al. 2022 ), release 214.The tree was pruned using Ne wic k utilities (Junier and Zdobnov 2010 ) to k ee p onl y the subtr ee encompassing the last common ancestor (LCA) of the DIG classes (as defined by GTDB) Legionellales , Coxiellales , DSM1500 ( Aquicella spp.), Diplorickettsiales , Francisellales , Berkiellales , and Piscirickettsiales .All 337 descendants of the DIG LCA were k e pt, but larger groups were collapsed for readability.On the tree, classes are highlighted according to their placement in GTDB.

Figure 3 .
Figure 3. Geogr a phical distribution of DIGs in various envir onments.Eac h panel r epr esents one of the five selected DIG families.The sampling location is r epr esented with a dot.Samples ar e colour ed according to the biome of origin.(A) DIG, (B) Coxiellaceae , (C) Legionellaceae , (D) Piscirickettsiaceae , (E) Francisellaceae , and (F) Fastidiosibacteraceae .
(v an Sc haik et al. 2013 ), Ric kettsiella(Bouc hon et al. 2011 ), and Legionella(Segal et al. 2005, O'Connor et al. 2011 ) with their hosts, by injecting a variety of protein effectors (see section below) into the host cell.Within the host cell, Dot/Icm substrates have been shown to manipulate various host cellular processes, enabling the

Table 1 .
Nonexhaustive list of taxa in the group Deep Intracellular Gammaproteobacteria and their related host/environment.